The hepatocyte possesses numerous physiological functions, including a very important function associated with the metabolism of what is called xenobiotics, wherein drugs, food additives, environmental pollutants and other xenobiotics are metabolized to ready-to-excrete forms. As such, the xenobiotic-metabolizing function sometimes also leads to mutagenesis, toxicity manifestation or substance efficacy manifestation by xenobiotics, and is under very extensive research. For this reason, cultured hepatocytes have been deemed not only to serve as a substitute for laboratory animals, as well as a quick, inexpensive and accurate test method for investigating metabolism in the liver, but also to enable the preparation of what is called artificial liver to substitute for hepatic functions.
However, human normal hepatocytes as isolated from living tissue cannot be subcultured. Cells which can be established as cell cultures often lack the essential differentiating characters; the resulting cell culture often does not accurately reflect the functions of the tissue to which they essentially belong. The class of enzymes involved in the metabolism of what is called xenobiotics in hepatocytes, in particular, lose their activity in a very short time in primary culture; no established cells have been found to sufficiently have the essential characters (J. Dich et al., Hepatology, 8, 39–45 (1988)). Against this background, there has been a wide demand for hepatocytes which have the capability of metabolizing xenobiotics and which permit cultivation. A cell culture of the human liver is prepared by selecting human tumor cells and exemplified by HepG2 (Aden et al., Nature, 282, 615–616, 1979). However, these cells are of tumor cell origin and do not represent immortalized normal cells. To immortalize normal cells, i.e., to allow normal cells to proliferate limitlessly, introduction of the T antigen gene of SV (simian virus) 40 origin, for example, is commonly available. However, no immortalized cell cultures of human hepatic normal parenchymal origin are known to allow observation of the immortalization of normal parenchymal cells of the liver, more specifically enzyme activity involved in the metabolism of xenobiotics, the expression of a gene encoding an enzyme involved in the metabolism of xenobiotics, or the induction of expression of a gene encoding an enzyme involved in the metabolism of xenobiotics. In addition, serum components are essential to media for cultivation of a large number of established cells. This necessity of serum components has been problematic in that not only the stability of cultured cell properties is considerably impeded due to a lack of the qualitative stability of the serum but also the stable, accurate and inexpensive use of established cells is considerably hampered due to the very high price of the serum. Accordingly, proliferation of an established immortalized cell culture in a serum-free medium, while stably retaining its character, would be industrially very beneficial.